PROPOSAL SUMMARY Excessive or inappropriate fear responses are a hallmark of numerous psychiatric disorders, including Post- Traumatic Stress Disorder (PTSD) and anxiety. While research has made critical progress in understanding fear neural circuitry, there is still much unknown. Further, available treatments for such disorders vary greatly in their effectiveness for individuals and lasting outcomes. The long-term goal of this research is to deepen the understanding of the neural circuits underlying learned fears in males and females and provide basic research that may inform the development of novel treatments. The objective of the proposed research training plan is to uncover a ventrolateral periaqueductal grey (vlPAG) to the medial subdivision of the central amygdala (CeM) neural circuit responsible for updating future fear to uncertain/unpredictable cues via prediction error signaling. Prediction errors are learning signals that are generated when there is a discrepancy between a predicted and received outcome. Using optogenetics, preliminary findings indicate vlPAG activity is necessary for aversive positive prediction errors (+PEs), which act to increase fear to a predictive cue upon future encounters. It is still unknown, however, where the vlPAG sends +PE signals in order to update future fear expectancies. The aims of this proposal are designed specifically to identify the brain region receiving vlPAG +PEs. The vlPAG directly projects to CeM, an area essential to fear learning and plasticity. It is hypothesized that a vlPAG-CeM circuit updates future fear via +PE signals. Aim 1 will use an optogenetic approach in rats to inhibit vlPAG terminals in the CeM precisely at the time of +PE (shock) during an innovative fear discrimination paradigm designed to generate prediction errors. It is expected that inhibition of these terminals will prevent +PE increases in fear, thereby decreasing fear to an uncertain cue. Aim 2 will then demonstrate a causal role for the vlPAG in CeM prediction error-related signaling by recording CeM single-unit activity during fear discrimination, including +PE (shock) periods, and during optogenetic inhibition of the vlPAG. It is predicted that CeM neurons will show increased firing after surprising foot shock, which will track with behavioral fear updating. VlPAG inhibition at the time of +PE will abolish CeM +PE-related signaling and its relationship to fear updating. These independent yet complementary aims will provide critical new insights into neural circuits for prediction error signaling and fear expression. Manipulation of aversive prediction errors could be an effective, innovative means of reducing excessive/inappropriate fear responses related to PTSD and anxiety.